This project seeks to elucidate mechanisms by which diabetes alters retinal endothelial cell barrier function and survival and to define the role of vascular endothelial cell growth factor (VEGF) in the pathology. Expression of VEGF and its receptor, VEGFR2, is increased in diabetic retinopathy. Paradoxically, while VEGF is a potent survival factor, retinal capillary dropout accompanies the diabetes-induced increases in its expression. Moreover, even though VEGF and VEGFR2 are abundant in normal adult retinas, blood-retinal barrier function remains normal. Thus, increased VEGF/VEGFR2 expression does not fully explain the diabetes-induced vascular alterations. Preliminary data show that diabetes and high glucose cause increases in retinal nitric oxide synthase (NOS) expression and activity and in formation of nitric oxide (NO), superoxide (O2-) and their product, peroxynitrite (ONOO-). These reactive oxygen species (ROS) are known to alter intracellular signaling functions. Preliminary data suggest that VEGF increases paracellular permeability by a process involving activation and nuclear translocation of beta-catenin, leading to activation of urokinase plasminogen activator receptor (uPAR) and decreases in the tight junction protein occludin. In stable monolayers, beta-catenin promotes cell adhesion by linking vascular endothelial cadherin to the actin cytoskeleton, but on growth factor stimulation beta-catenin binds and activates the transcription factor LEF-1 (lymphocyte enhancer factor-1) leading to uPAR expression. Beta-catenin activation by VEGF involves an interaction with phosphatidylinositol (PI)-3 kinase, which promotes cytosolic accumulation of free beta-catenin by inhibiting its degradation by glycogen synthase kinase 3. PI-3 kinase promotes cell migration and survival and is a target for protein tyrosine nitration by ONOO-. Beta-catenin/LEF-1 binding is enhanced by NO. Both NO and O2- inhibit tyrosine phosphatases. Our working model is that the effects of VEGF increasing endothelial cell permeability and promoting cell survival are normally tightly coupled by the coordinated function of the beta-catenin and PI-3 kinase signaling pathways. In diabetes the permeability and survival functions of VEGF are uncoupled due to the effects of NO, O2- and ONOO- in altering the beta-catenin and PI-3 kinase signaling pathways. This will be tested by experiments using a combination of cellular and molecular analytical approaches to test the following hypotheses: 1) VEGF promotes an increase in paracellular permeability by a process involving beta-catenin and PI-3 kinase mediated increases in uPAR. 2) High glucose increases permeability and reduces survival by increasing ROS formation and altering VEGF, beta-catenin and PI-3 kinase intracellular signaling. 3) Diabetes increases vascular permeability and causes vascular cell death by increasing ROS formation.